Multiomics Characterization of GCSH + Macrophages Reveals Therapeutic Vulnerabilities and Immune-Metabolic Crosstalk in Triple-Negative Breast Cancer.

Background: Tumor-associated macrophages (TAMs) are key regulators of immune homeostasis within the tumor microenvironment (TME) and play critical roles in malignant progression. However, the molecular mechanisms linking macrophage metabolic remodeling to immune regulation remain incompletely understood. Glycine cleavage system H protein (GCSH), a core regulator of copper-dependent cell death, has been implicated in metabolic regulation in triple-negative breast cancer (TNBC), suggesting a potential role in macrophage-mediated TME remodeling. Methods: We integrated single-cell RNA sequencing and spatial transcriptomic data from TNBC tissues to systematically characterize macrophage subpopulations with high GCSH expression. Pseudotime trajectory analysis, cuproptosis-related scoring, cell-cell communication inference, metabolic pathway enrichment, and spatial localization analyses were performed to delineate their functional heterogeneity and microenvironmental context. In addition, mutation profiling, immunogenomic analysis, drug sensitivity prediction, and in vitro and in vivo functional experiments were conducted to comprehensively evaluate the biological and therapeutic relevance of GCSH. Results: GCSH expression was predominantly enriched in macrophages, particularly in early activated subsets, and was associated with enhanced amino acid and lipid metabolic activity. GCSH + macrophages exhibited extensive interactions with T cells via pathways such as MIF-CD74-CXCR4 and LGALS9-CD45, contributing to an immunosuppressive, tumor-promoting microenvironment. Spatial analysis revealed their preferential localization at the tumor core-stroma interface. Notably, GCSH missense mutations were associated with increased M1 macrophage infiltration and enrichment of immune and inflammatory pathways. Clinically, high GCSH expression correlated with poor survival, genomic instability, and chemotherapy resistance. Functional experiments demonstrated that GCSH silencing suppressed tumor cell proliferation, migration, and clonogenicity, induced apoptosis, enhanced proinflammatory cytokine secretion, and significantly inhibited tumor growth in vivo. Conclusion: GCSH acts as a central molecular link between macrophage metabolic reprogramming, immune suppression, and TNBC progression, highlighting its potential as both a prognostic biomarker and therapeutic target.